Glycine Transporter-1 Inhibition Promotes Striatal Axon Sprouting Via NMDA Receptors in Dopamine Neurons

16778 • The Journal of Neuroscience, October 16, 2013 • 33(42):16778–16789 Development/Plasticity/Repair Glycine Transporter-1 Inhibition Promotes Striatal Axon Sprouting via NMDA Receptors in Dopamine Neurons Yvonne Schmitz,1 Candace Castagna,1 Ana Mrejeru,1 Jose´ E. Lizardi-Ortiz,1 Zoe Klein,1 Craig W. Lindsley,4 and David Sulzer1,2,3 1Departments of Neurology, 2Psychiatry, and 3Pharmacology, Columbia University Medical Center, New York, New York 10032, and 4Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, Tennessee 37232 NMDA receptor activity is involved in shaping synaptic connections throughout development and adulthood. We recently reported that briefactivationofNMDAreceptorsonculturedventralmidbraindopamineneuronsenhancedtheiraxongrowthrateandinducedaxonal branching. To test whether this mechanism was relevant to axon regrowth in adult animals, we examined the reinnervation of dorsal striatum following nigral dopamine neuron loss induced by unilateral intrastriatal injections of the toxin 6-hydroxydopamine. We used a pharmacological approach to enhance NMDA receptor-dependent signaling by treatment with an inhibitor of glycine transporter-1 that elevates levels of extracellular glycine, a coagonist required for NMDA receptor activation. All mice displayed sprouting of dopaminergic axons from spared fibers in the ventral striatum to the denervated dorsal striatum at 7 weeks post-lesion, but the reinnervation in mice treated for 4 weeks with glycine uptake inhibitor was approximately twice as dense as in untreated mice. The treated mice also displayed higher levels of striatal dopamine and a complete recovery from lateralization in a test of sensorimotor behavior. We confirmed that the actions of glycine uptake inhibition on reinnervation and behavioral recovery required NMDA receptors in dopamine neurons using targeted deletion of the NR1 NMDA receptor subunit in dopamine neurons. Glycine transport inhibitors promote functionally relevant sprouting of surviving dopamine axons and could provide clinical treatment for disorders such as Parkinson’s disease. Introduction 2011) and mediate growth cone turning in response to glutamate During development and in adulthood, NMDA glutamate recep- gradients (Zheng et al., 1996). We recently reported that a brief tor activity is involved in synapse elimination or stabilization, exposure to NMDA receptor agonists enhanced axonal growth and inhibition or promotion of axonal sprouting (Li et al., 1994; rate and branching in cultured dopaminergic midbrain neurons Katz and Shatz, 1996; Constantine-Paton and Cline, 1998; (Schmitz et al., 2009) consistent with prior studies on cerebellar Ruthazer and Cline, 2004; Colonnese et al., 2005; Lee et al., 2005). granule cells (Pearce et al., 1987; Rashid and Cambray-Deakin, The roles of axonal presynaptic versus somatodendritic postsynaptic 1992). Here, we tested the hypothesis that NMDA receptor activ- NMDA receptors in these processes are not well understood. Presyn- ity promotes sprouting of dopaminergic axons in vivo by study- aptic NMDA receptor expression on axons appears to be high during ing sprouting from spared fibers in the ventral striatum to the early development and drops drastically in adulthood (Herkert et al., dorsal striatum following striatal 6-hydroxydopamine (6- 1998; Lien et al., 2006; Corlew et al., 2007; Wang et al., 2011). The OHDA)-induced lesions in mature mice. Lesions were adjusted functional relevance of presynaptic NMDA receptors is controver- so that most cells in the substantia nigra (SNpc) innervating the sial (Christie and Jahr, 2008; Pugh and Jahr, 2011), although several dorsal striatum were lost, but cells in the ventral tegmental area studies suggest a modulatory effect on transmitter release (Tzingou- (VTA) innervating the ventral striatum were spared. This lesion nis and Nicoll, 2004; Larsen et al., 2011). model mimics the denervation pattern found in brains of patients In cultured neurons, NMDA receptors tend to be expressed in with Parkinson’s disease, where the dopaminergic innervation of axons and axonal growth cones (Schmitz et al., 2009; Wang et al., the lateral putamen is lost while that of the most medial portion of the putamen, the caudate, and nucleus accumbens remains Received June 26, 2012; revised Sept. 10, 2013; accepted Sept. 13, 2013. relatively intact (Miller et al., 1999). Author contributions: Y.S. and D.S. designed research; Y.S., C.C., A.M., J.E.L.-O., and Z.K. performed research; To enhance NMDA receptor activity pharmacologically, we C.W.L. contributed unpublished reagents/analytic tools; Y.S. analyzed data; Y.S. and D.S. wrote the paper. used an uptake inhibitor of the amino acid glycine, which is a This work was supported by the JPB Foundation, the Michael J Fox Foundation, the National Parkinson’s Foun- coagonist that binds to the NR1 subunit and is required for re- dation, the Parkinson’s Disease Foundation (PDF), and the Udall Center of Excellence at Columbia University. Z.K. received a summer student fellowship from PDF. We are grateful to Tinmarla Oo, Tatyana Kareva, and Robert Burke ceptor activation (Clements and Westbrook, 1991; Berger et al., for training and advice in stereotactic 6-OHDA injections; to Shane Grealish and Eilis Dowd for advice on behavioral 1998). Glycine transporter 1 (GlyT1) is widely expressed in the tests; to Eugene Mosharov for help with HPLC; and to Paul Witkovsky for valuable comments on this manuscript. forebrain in glial as well as neuronal cells (Smith et al., 1992; The authors declare no competing financial interests. Raiteri and Raiteri, 2010) and has been shown to regulate glycine CorrespondenceshouldbeaddressedtoeitherYvonneSchmitzorDavidSulzer,DepartmentofNeurology,650W 168th Street BB308, New York, NY 10032. E-mail: [email protected] or [email protected]. occupancy of NMDA receptors in the CNS (Berger et al., 1998; DOI:10.1523/JNEUROSCI.3041-12.2013 Bergeron et al., 1998) leading to enhanced NMDA currents and Copyright © 2013 the authors 0270-6474/13/3316778-12$15.00/0 LTP in hippocampal CA1 (Martina et al., 2004). Importantly, Schmitz et al. • GlyT1 Inhibitor Promotes Dopaminergic Axon Growth J. Neurosci., October 16, 2013 • 33(42):16778–16789 • 16779 GlyT1 inhibitors increase glycine levels in the mouse striatum Olympus IX81 microscope using MetaMorph software (Molecular De- threefold (Alberati et al., 2012). GlyT1 inhibitors have been ex- vices). Images of sections immunostained for TH from mice expressing plored for potential treatment of NMDA receptor hypofunction tdTomato (cross of tdTomato-ROSA reporter mice with DATCre mice) in schizophrenia (Bridges et al., 2008; Javitt, 2008). Here we re- were taken on a Leica DM6000 confocal microscope. port that the GlyT1 inhibitor, ACPPB (Lindsley et al., 2006; To estimate the lesion size in the SNpc and VTA every second section through the midbrain was collected and immunolabeled for TH. Five Wolkenberg et al., 2009), promoted functional dopaminergic re- sections containing SNpc and three to four sections containing VTA innervation of the 6-OHDA-lesioned dorsal striatum in mature (between bregma –2.92 and Ϫ3.16 mm; Franklin and Paxinos, 1997) mice and that this action depended on NMDA receptors ex- were analyzed. Using MetaMorph software, a region around the area pressed by dopaminergic neurons. containing SNpc and VTA, respectively, was drawn and the image threshold was set above the fluorescence of the cortex. The size of the area Materials and Methods covered by labeled cells was expressed as the percentage of the area size in Mice. Mice were kept according to National Institutes of Health guide- the unlesioned hemisphere. All mice included in the analysis had SNpc lines under a 12 h light/dark cycle with ad libitum access to food and lesions Ͼ70% and VTA lesions smaller than 50% (see Fig. 1E,F). water. Some mice were kept under mild food restriction for up to 4 d (see Fiber density in the striatum was assessed in 12–15 adjacent cryosec- below, Behavioral tests). All animal studies were reviewed and approved tions immunolabeled for the dopamine transporter (between bregma 2 by the Institutional Animal Care and Use Committee at Columbia Uni- 1.18 and 0.82 mm). In each section, four regions of interest (0.12 mm ) versity Medical Center. C57BL/6 mice and Slc6a3 Cre mice, used for the arranged dorsoventrally in the center of the section were analyzed with conditional inactivation of Grin1, were obtained from The Jackson Lab- the most dorsal directly under the corpus callosum and the most ventral oratory. Grin1loxP mice were kindly provided by the laboratory of Dr. region overlapping with part of the spared ventral striatum (see Fig. 2A). Charles Inturrisi (Cornell University, New York) with the permission of The threshold was set in the center of each region so that the fiber bundles Dr. Susumu Tonegawa. Mice used in this study were backcrossed with that cross the striatum were excluded. The threshold area per region on C57BL/6 mice for at least six generations. All mice were male and be- the lesioned side was expressed as percentage of the threshold area per tween 3 and 5 months old at the time of 6-OHDA injections. region in the respective control hemisphere and the average for each For the conditional inactivation of Grin1, the gene that encodes the region in the 12–15 sections per mouse was calculated (Fig. 2A; see the NR1 subunit that is contained in all NMDA receptors, a transgenic line, examples of thresholding). The initial set

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